In the field of

genetic sequencing Genetic Sequencing may refer to: * DNA sequencing * Whole genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entiret ...

, genotyping by sequencing, also called GBS, is a method to discover

single nucleotide polymorphisms In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...

(SNP) in order to perform

genotyping Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. ...

studies, such as genome-wide association studies ( GWAS). GBS uses

restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...

s to reduce genome complexity and genotype multiple DNA samples. After digestion,

PCR PCR or pcr may refer to: Science * Phosphocreatine, a phosphorylated creatine molecule * Principal component regression, a statistical technique Medicine * Polymerase chain reaction ** COVID-19 testing, often performed using the polymerase chain r ...

is performed to increase fragments pool and then GBS libraries are sequenced using next generation sequencing technologies, usually resulting in about 100bp single-end reads. It is relatively inexpensive and has been used in

plant breeding Plant breeding is the science of changing the traits of plants in order to produce desired characteristics. It has been used to improve the quality of nutrition in products for humans and animals. The goals of plant breeding are to produce cr ...

. Although GBS presents an approach similar to restriction-site-associated DNA sequencing (RAD-seq) method, they differ in some substantial ways.

Methods

GBS is a robust, simple, and affordable procedure for SNP discovery and mapping. Overall, this approach reduces genome complexity with restriction enzymes (REs) in high-diversity, large genomes species for efficient high-throughput, highly multiplexed sequencing. By using appropriate REs, repetitive regions of genomes can be avoided and lower copy regions can be targeted, which reduces alignments problems in genetically highly diverse species. The method was first described by Elshire et al. (2011). In summary, high

molecular weight A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...

DNAs are extracted and digested using a specific RE previously defined by cutting frequently in the major repetitive fraction of the genome. ''ApeKI'' is the most used RE. Barcode adapters are then ligated to

sticky ends DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...

and

PCR amplification The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...

is performed. Next-generation sequencing technology is performed resulting in about 100 bp single-end reads. Raw sequence data are filtered and aligned to a reference genome using usually Burrows–Wheeler alignment tool (BWA) or Bowtie 2. The next step is to identify SNPs from aligned tags and score all discovered SNPs for various coverage, depth and genotypic statistics. Once a large-scale, species-wide SNP production has been run, it is possible to quickly call known SNPs in newly sequenced samples. When initially developed, the GBS approach was tested and validated in recombinant inbred lines (RILs) from a high-resolution maize mapping population (IBM) and doubled haploid (DH) barley lines from the Oregon Wolfe Barley (OWB) mapping population. Up to 96 RE (ApeKI)-digested DNA samples were pooled and processed simultaneously during the GBS library construction, which was checked on a Genome Analyzer II (Illumina, Inc.). Overall, 25,185 biallelic tags were mapped in maize, while 24,186 sequence tags were mapped in barley. Barley GBS marker validation using a single DH line (OWB003) showed 99% agreement between the reference markers and the mapped GBS reads. Although barley lacks a complete genome sequence, GBS does not require a reference genome for sequence tag mapping, the reference is developed during the process of sampling genotyping. Tags can also be treated as dominant markers for alternative genetic analysis in the absence of a reference genome. Other than the multiplex GBS skimming, imputation of missing SNPs has the potential to further reduce GBS costs. GBS is a versatile and cost-effective procedure that will allow mining genomes of any species without prior knowledge of its genome structure.

References

{{Reflist Genetics Biotechnology DNA sequencing

Methods

See also

References